1. Field of the Invention
The present invention concerns a process for the simultaneous purification and concentration of an enzyme, consisting in dextran-sucrase, from the supernatant phase of the culture of the enzyme-producing bacteria.
The dextrans are glucose polymers that are obtained by enzymatic transformation of sucrose. These polysaccharides have various industrial applications depending on their molecular weight. There can be cited as examples the utilization of high molecular weight dextrans in the petroleum industry as viscosigenous agent, the utilization of dextrans of average molecular weight in the foodstuffs industry and that of low molecular weight dextrans in the pharmaceutical industry.
Numerous bacterial layers forming part of the Lactobacillus, Streptococcus or Leuconostoc species possess a dextran-sucrase activity. Among these, however, the strain of Leuconostoc mesenteroides NRRL B 512 (F), that excretes the dextran-sucrase enzyme, when it is cultivated in the presence of sucrose, presents particularly interesting stability properties, an important productivity and a non-pathogenic character, that have allowed it to be utilized on an industrial scale. Furthermore, the dextran-sucrase obtained from this bacterial strain produces an only slightly branched linear dextran (95% .alpha.1.fwdarw.6 glycosidic bonds) which is soluble in water, qualities which render it a very worthwhile product on the industrial scale.
Current industrial processes of enzymatic synthesis of dextrans in which the dextran-sucrase of Leuconostoc mesenteroides B 512 (F) is used present, however, drawbacks.
Indeed, the industrial process consists in producing the dextran-sucrase enzyme in very large size fermentation tanks. Once the enzyme has been excreted by the bacteria, L. mesenteroides NRRL B 512 (F), slightly or not purified, it is contacted with its substrate, sucrose, and there is thus produced crude dextran of which the conditions of synthesis cannot be efficiently controlled. This dextran is thereafter subjected to various physico-chemical treatments for its recovery and purification; the overall yield of the reaction is thus relatively low.
The present invention concerns an economical purification process of the dextran-sucrase enzyme of Leuconostoc-mesenteroides, able, furthermore, to be easily integrated in an on line production process of the enzyme. The high productivity thus obtained associated with a simple and remarkably efficient purification technique, which is the object of the present invention, justifies the utilization of this enzymatic preparation on the industrial scale and allows, furthermore, through a precise control of the synthesis conditions, the obtention of various types of dextrans according to the field of application envisaged.
Recent developments of enzymatic synthesis have led researchers to take on interest in the problems of purification of enzymes. Numerous purification techniques are utilized with this aim, and by way of example can be cited filtration and its micro- and ultra-filtration embodiments, centrifugation, chromatography, precipitation, liquid-liquid extraction, which has recently been the object of developments involving the phenomenon known as "phase demixing". In this respect there can be cited the works of M. R. KULA (Applied Biochemistry and Bioengineering Vol. 2 pp 71-95 Academic Press 1977 and U.S. Pat. No. 4,144,130) that describe a technique for the purification of various enzymes operating in the phase demixing between two polymers, the polyethylenglycol and the dextran that, at certain concentrations in aqueous solution, become incompatible, thus provoking the appearance of two distinct liquid phases between which are divided the substances present in the medium in function of their solubility of their affinity for one or other of the two phases in the operating conditions.
The culture medium of the strain producing the dextran sucrase contains other extra cellular enzymes that it is important to be able to eliminate. In particular, two saccharolytic enzymes able to utilize the sucrose are excreted by Leuconostoc measenteroides type strains levan-sucrase and invertase. These two enzymes are particularly harmful; on the one hand they reduce the dextran yield, on the other hand, they prevent the satisfactory control of the dextran production. These two contaminating activities can be schematized as follows: ##STR1##
The presence of glucose reveals a contaminating activity; indeed, a pure solution of dextran-sucrase in the presence of sucrose does not produce glucose in the free form. The analysis of the relative percentage of glucose and fructose appearing during purification upon measurement of the dextran-sucrase activity is thus a criterion of the purity of the enzyme.
The object of the present invention is to overcome the previous drawbacks by providing an efficient, simple and economic process of simultaneous purification and concentration of dextran-sucrase operating in an original manner the liquid-liquid extraction by phase demixing.
The present invention concerns a process for the simultaneous purification and concentration of the dextran-sucrase enzyme from the culture medium on sucrose of the Leuconostoc mesenteroides bacteria producing the extra cellular enzyme, the said medium containing dextran, in which, during a first step, an aqueous solution of a polyether is added to the medium in such a quantity such that in the medium, two non-miscible phases appear and said medium is maintained under stirring in order to ensure close and prolonged contact between the two polymers and the biochemical substances; then, in a second step, the lower dextran-rich phase of the medium is separated from the upper polyether-rich phase, the lower phase constituting the dextran-sucrase enriched enzymatic preparation.
The process is expressed by a separation in two phases of the culture medium:
a heavy dextran-rich phase that contains the concentrated and purified dextran-sucrase enzyme with a partition coefficient ##EQU1## close to zero; a lighter polyether-rich phase that contains impurities, especially proteins and sugars as well as the contaminating enzymatic activities, phase that is eliminated. By way of usable polyethers in the process according to the PA1 polyethylenglycol (PEG), PA1 polypropylenglycol, PA1 methoxypolyethylenglycol PA1 polyethylenglycol trimethylamine, PA1 polyethylenglycol sulfonate etc. . . PA1 F: fructose PA1 levan-sucrase activity (ULS): total glucose released PA1 dextran-sucrase activity (UDS): total fructose released PA1 total saccharolytic activity: fructose+glucose.
invention can be cited:
and their derivates such as:
The average molecular weight of the polyether utilized will generally be between 400 and 20,000. This molecular weight will be chosen in function of the nature of the dextran present in the medium.
The quantity of polyether added will generally be such that the amount in the medium of polyalcohol after addition thereof will be between about 1 and 15% by weight. The optimal amount will be determined in function of the dextran content in the medium.
The process is operated substantially at ambient temperature, i.e. at a maximum temperature of about 30.degree. C. Furthermore, the pH of the medium will generally be between about 4.5 and 7.
It will be advantageous in order to facilitate the carrying out of the process to preferably eliminate, from the medium the cells, for example, by centrifugation.
From the works of M. R. KULA mentioned herein-above was shown that it was possible to purify enzymes by making use of the phase partition phenomenon. However, it appeared evident that during the purification process the enzymes are distributed between the two phases but that the purified enzyme fraction is obtained solely from the upper PEG phase, the lower dextran phase acting essentially to collect the cellular debris and impurities present in the medium.
Now, it has been observed by the applicant that in the process according to the invention, the quasi totality of the dextran-sucrase enzyme is carried along in the lower dextran phase whereas the other contaminating enzymatic activities preferably pass into the upper polyether phase. The lower dextran phase can also act as a source of dextran-sucrase enzymatic preparations and that as purification is accompanied by a very high concentration, the volume of the lower phase corresponding to several % of the volume of the medium subjected to the phase partition.
Of course, the process can be carried out in two or several steps during which the lower phase issued from the preceding step will be subjected to a further extraction by phase partition under analogous operating conditions, after having, where necessary, slightly diluted it in order to facilitate the process.
This purification process on several steps can be realized in line in a liquid-liquid extraction column operating at counter-current.
The process of the invention is illustrated by the examples given herein-below, which are to be interpreted at the light of the following definitions.